Belay T. Ayele

Potential sites of bioactive gibberellin production during reproductive growth in Arabidopsis.

Gibberellin 3-oxidase (GA3ox) catalyzes the final step in the synthesis of bioactive gibberellins (GAs). We examined the expression patterns of all four GA3ox genes in Arabidopsis thaliana by promoter–β-glucuronidase gene fusions and by quantitative RT-PCR and defined their physiological roles by characterizing single, double, and triple mutants. In developing flowers, GA3ox genes are only expressed in stamen filaments, anthers, and flower receptacles. Mutant plants that lack both GA3ox1 and GA3ox3 functions displayed stamen and petal defects, indicating that these two genes are important for GA production in the flower. Our data suggest that de novo synthesis of active GAs is necessary for stamen development in early flowers and that bioactive GAs made in the stamens and/or flower receptacles are transported to petals to promote their growth. In developing siliques, GA3ox1 is mainly expressed in the replums, funiculi, and the silique receptacles, whereas the other GA3ox genes are only expressed in developing seeds. Active GAs appear to be transported from the seed endosperm to the surrounding maternal tissues where they promote growth. The immediate upregulation of GA3ox1 and GA3ox4 after anthesis suggests that pollination and/or fertilization is a prerequisite for de novo GA biosynthesis in fruit, which in turn promotes initial elongation of the silique.

Developmental and Hormonal Regulation of Gibberellin Biosynthesis and Catabolism in Pea Fruit

In pea (Pisum sativum), normal fruit growth requires the presence of the seeds. The coordination of growth between the seed and ovary tissues involves phytohormones; however, the specific mechanisms remain speculative. This study further explores the roles of the gibberellin (GA) biosynthesis and catabolism genes during pollination and fruit development and in seed and auxin regulation of pericarp growth. Pollination and fertilization events not only increase pericarp PsGA3ox1 message levels (codes for GA 3-oxidase that converts GA20 to bioactive GA1) but also reduce pericarp PsGA2ox1 mRNA levels (codes for GA 2-oxidase that mainly catabolizes GA20 to GA29), suggesting a concerted regulation to increase levels of bioactive GA1 following these events. 4-Chloroindole-3-acetic acid (4-Cl-IAA) was found to mimic the seeds in the stimulation of PsGA3ox1 and the repression of PsGA2ox1 mRNA levels as well as the stimulation of PsGA2ox2 mRNA levels (codes for GA 2-oxidase that mainly catabolizes GA1 to GA8) in pericarp at 2 to 3 d after anthesis, while the other endogenous pea auxin, IAA, did not. This GA gene expression profile suggests that both seeds and 4-Cl-IAA can stimulate the production, as well as modulate the half-life, of bioactive GA1, leading to initial fruit set and subsequent growth and development of the ovary. Consistent with these gene expression profiles, deseeded pericarps converted [14C]GA12 to [14C]GA1 only if treated with 4-Cl-IAA. These data further support the hypothesis that 4-Cl-IAA produced in the seeds is transported to the pericarp, where it differentially regulates the expression of pericarp GA biosynthesis and catabolism genes to modulate the level of bioactive GA1 required for initial fruit set and growth.

Regulation of Wheat Seed Dormancy by After-Ripening Is Mediated by Specific Transcriptional Switches That Induce Changes in Seed Hormone Metabolism and Signaling

Treatments that promote dormancy release are often correlated with changes in seed hormone content and/or sensitivity. To understand the molecular mechanisms underlying the role of after-ripening (seed dry storage) in triggering hormone related changes and dormancy decay in wheat (Triticum aestivum), temporal expression patterns of genes related to abscisic acid (ABA), gibberellin (GA), jasmonate and indole acetic acid (IAA) metabolism and signaling, and levels of the respective hormones were examined in dormant and after-ripened seeds in both dry and imbibed states. After-ripening mediated developmental switch from dormancy to germination appears to be associated with declines in seed sensitivity to ABA and IAA, which are mediated by transcriptional repressions of PROTEIN PHOSPHATASE 2C, SNF1-RELATED PROTEIN KINASE2, ABA INSENSITIVE5 and LIPID PHOSPHATE PHOSPHTASE2, and AUXIN RESPONSE FACTOR and RELATED TO UBIQUITIN1 genes. Transcriptomic analysis of wheat seed responsiveness to ABA suggests that ABA inhibits the germination of wheat seeds partly by repressing the transcription of genes related to chromatin assembly and cell wall modification, and activating that of GA catabolic genes. After-ripening induced seed dormancy decay in wheat is also associated with the modulation of seed IAA and jasmonate contents. Transcriptional control of members of the ALLENE OXIDE SYNTHASE, 3-KETOACYL COENZYME A THIOLASE, LIPOXYGENASE and 12-OXOPHYTODIENOATE REDUCTASE gene families appears to regulate seed jasmonate levels. Changes in the expression of GA biosynthesis genes, GA 20-OXIDASE and GA 3-OXIDASE, in response to after-ripening implicate this hormone in enhancing dormancy release and germination. These findings have important implications in the dissection of molecular mechanisms underlying regulation of seed dormancy in cereals.

Genome-wide Comparative Analysis of Annexin Superfamily in Plants

Most annexins are calcium-dependent, phospholipid-binding proteins with suggested functions in response to environmental stresses and signaling during plant growth and development. They have previously been identified and characterized in Arabidopsis and rice, and constitute a multigene family in plants. In this study, we performed a comparative analysis of annexin gene families in the sequenced genomes of Viridiplantae ranging from unicellular green algae to multicellular plants, and identified 149 genes. Phylogenetic studies of these deduced annexins classified them into nine different arbitrary groups. The occurrence and distribution of bona fide type II calcium binding sites within the four annexin domains were found to be different in each of these groups. Analysis of chromosomal distribution of annexin genes in rice, Arabidopsis and poplar revealed their localization on various chromosomes with some members also found on duplicated chromosomal segments leading to gene family expansion. Analysis of gene structure suggests sequential or differential loss of introns during the evolution of land plant annexin genes. Intron positions and phases are well conserved in annexin genes from representative genomes ranging from Physcomitrella to higher plants. The occurrence of alternative motifs such as K/R/HGD was found to be overlapping or at the mutated regions of the type II calcium binding sites indicating potential functional divergence in certain plant annexins. This study provides a basis for further functional analysis and characterization of annexin multigene families in the plant lineage.

Transcriptional programs regulating seed dormancy and its release by after‐ripening in common wheat (Triticum aestivum L.)

Seed dormancy is an important agronomic trait in wheat (Trticum aestivum). Seeds can be released from a physiologically dormant state by after-ripening. To understand the molecular mechanisms underlying the role of after-ripening in conferring developmental switches from dormancy to germination in wheat seeds, we performed comparative transcriptomic analyses between dormant (D) and after-ripened (AR) seeds in both dry and imbibed states. Transcriptional activation of genes represented by a core of 22 and 435 probesets was evident in the dry and imbibed states of D seeds, respectively. Furthermore, two-way ANOVA analysis identified 36 probesets as specifically regulated by dormancy. These data suggest that biological functions associated with these genes are involved in the maintenance of seed dormancy. Expression of genes encoding protein synthesis/activity inhibitors was significantly repressed during after-ripening, leading to dormancy decay. Imbibing AR seeds led to transcriptional activation of distinct biological processes, including those related to DNA replication, nitrogen metabolism, cytoplasmic membrane-bound vesicle, jasmonate biosynthesis and cell wall modification. These after-ripening-mediated transcriptional programs appear to be regulated by epigenetic mechanisms. Clustering of our microarray data produced 16 gene clusters; dormancy-specific probesets and abscisic acid (ABA)-responsive elements were significantly overrepresented in two clusters, indicating the linkage of dormancy in wheat with that of seed sensitivity to ABA. The role of ABA signalling in regulating wheat seed dormancy was further supported by the down-regulation of ABA response-related probesets in AR seeds and absence of differential expression of ABA metabolic genes between D and AR seeds.

Hemoglobin Control of Cell Survival/Death Decision Regulates in Vitro Plant Embryogenesis

Hemoglobins modulate embryogenesis by regulating programmed cell death. Programmed cell death (PCD) in multicellular organisms is a vital process in growth, development, and stress responses that contributes to the formation of tissues and organs. Although numerous studies have defined the molecular participants in apoptotic and PCD cascades, successful identification of early master regulators that target specific cells to live or die is limited. Using Zea mays somatic embryogenesis as a model system, we report that the expressions of two plant hemoglobin (Hb) genes (ZmHb1 and ZmHb2) regulate the cell survival/death decision that influences somatic embryogenesis through their cell-specific localization patterns. Suppression of either of the two ZmHbs is sufficient to induce PCD through a pathway initiated by elevated NO and Zn2+ levels and mediated by production of reactive oxygen species. The effect of the death program on the fate of the developing embryos is dependent on the localization patterns of the two ZmHbs. During somatic embryogenesis, ZmHb2 transcripts are restricted to a few cells anchoring the embryos to the subtending embryogenic tissue, whereas ZmHb1 transcripts extend to several embryonic domains. Suppression of ZmHb2 induces PCD in the anchoring cells, allowing the embryos to develop further, whereas suppression of ZmHb1 results in massive PCD, leading to abortion. We conclude that regulation of the expression of these ZmHbs has the capability to determine the developmental fate of the embryogenic tissue during somatic embryogenesis through their effect on PCD. This unique regulation might have implications for development and differentiation in other species.

Integrated analysis of seed proteome and mRNA oxidation reveals distinct post‐transcriptional features regulating dormancy in wheat (Triticum aestivum L.)

Wheat seeds can be released from a dormant state by after-ripening; however, the underlying molecular mechanisms are still mostly unknown. We previously identified transcriptional programmes involved in the regulation of after-ripening-mediated seed dormancy decay in wheat (Triticum aestivum L.). Here, we show that seed dormancy maintenance and its release by dry after-ripening in wheat is associated with oxidative modification of distinct seed-stored mRNAs that mainly correspond to oxidative phosphorylation, ribosome biogenesis, nutrient reservoir and α-amylase inhibitor activities, suggesting the significance of post-transcriptional repression of these biological processes in regulating seed dormancy. We further show that after-ripening induced seed dormancy release in wheat is mediated by differential expression of specific proteins in both dry and hydrated states, including those involved in proteolysis, cellular signalling, translation and energy metabolism. Among the genes corresponding to these proteins, the expression of those encoding α-amylase/trypsin inhibitor and starch synthase appears to be regulated by mRNA oxidation. Co-expression analysis of the probesets differentially expressed and oxidized during dry after-ripening along with those corresponding to proteins differentially regulated between dormant and after-ripened seeds produced three co-expressed gene clusters containing more candidate genes potentially involved in the regulation of seed dormancy in wheat. Two of the three clusters are enriched with elements that are either abscisic acid (ABA) responsive or recognized by ABA-regulated transcription factors, indicating the association between wheat seed dormancy and ABA sensitivity.

Functional genomics of seed dormancy in wheat: advances and prospects

Seed dormancy is a mechanism underlying the inability of viable seeds to germinate under optimal environmental conditions. To achieve rapid and uniform germination, wheat and other cereal crops have been selected against dormancy. As a result, most of the modern commercial cultivars have low level of seed dormancy and are susceptible to preharvest sprouting when wet and moist conditions occur prior to harvest. As it causes substantial loss in grain yield and quality, preharvest sprouting is an ever-present major constraint to the production of wheat. The significance of the problem emphasizes the need to incorporate an intermediate level of dormancy into elite wheat cultivars, and this requires detailed dissection of the mechanisms underlying the regulation of seed dormancy and preharvest sprouting. Seed dormancy research in wheat often involves after-ripening, a period of dry storage during which seeds lose dormancy, or comparative analysis of seeds derived from dormant and non-dormant cultivars. The increasing development in wheat genomic resources along with the application of transcriptomics, proteomics, and metabolomics approaches in studying wheat seed dormancy have extended our knowledge of the mechanisms acting at transcriptional and post-transcriptional levels. Recent progresses indicate that some of the molecular mechanisms are associated with hormonal pathways, epigenetic regulations, targeted oxidative modifications of seed mRNAs and proteins, redox regulation of seed protein thiols, and modulation of translational activities. Given that preharvest sprouting is closely associated with seed dormancy, these findings will significantly contribute to the designing of efficient strategies for breeding preharvest sprouting tolerant wheat.

Developmental and seed aging mediated regulation of antioxidative genes and differential expression of proteins during pre- and post-germinative phases in pea.

Enzymatic antioxidant system plays an important role in maintaining seed vigor and regulating plant growth and development. It involves a number of enzymes that scavenge excessive reactive oxygen species (ROS) produced during seed aging and also modulate the level of these compounds during plant developmental processes. This study investigated the transcriptional regulation of enzymatic antioxidative capacity in pea during the pre- and post-germinative phases and in response to seed aging by analyzing the spatio-temporal expression of five antioxidative genes: PsAPX, PsSOD, PsGRcyt, PsGRcm and PsCAT. Transcripts of all these genes were found in mature dry seeds, embryo axes and cotyledons of germinating seeds, and cotyledons, roots and shoots of young seedlings. However, PsAPX and PsSOD were predominant and exhibited developmental regulation, suggesting that these genes play important roles in controlling the intracellular homeostasis of ROS for promoting cell elongation, and thereby embryo axis expansion and early seedling growth in pea. Accelerated aging of pea seeds led to reduction in seed viability and seedling growth, and this effect was correlated with substantial decrease in the transcriptional activation of the prominent antioxidative genes. Furthermore, our proteomic analysis indicated the association of seed aging with changes in the abundance of specific proteins, revealing additional mechanisms underlying seed aging in pea.

Developmental and Embryo Axis Regulation of Gibberellin Biosynthesis during Germination and Young Seedling Growth of Pea

The expression patterns of five genes (PsGA20ox1, PsGA20ox2, PsGA3ox1, PsGA2ox1, and PsGA2ox2) encoding five regulatory gibberellin (GA) biosynthesis enzymes (two GA 20-oxidases, a GA 3β-hydroxylase, and two GA 2β-hydroxylases) were examined to gain insight into how these genes coordinate GA biosynthesis during germination and early postgermination stages of the large-seeded dicotyledonous plant pea (Pisum sativum). At the time the developing embryo fills the seed coat, high mRNA levels of PsGA20ox2 (primarily responsible for conversion of C20-GAs to GA20), PsGA2ox1 (primarily responsible for conversion of GA20 to GA29), and PsGA2ox2 (primarily responsible for conversion of GA1 to GA8) were detected in the seeds, along with high GA20 and GA29 levels, the enzymatic products of these genes. Embryo maturation was accompanied by a large reduction in PsGA20ox2 and PsGA2ox1 mRNA and lower GA20 and GA29 levels. However, PsGA2ox2 transcripts remained high. Following seed imbibition, GA20 levels in the cotyledons decreased, while PsGA3ox1 mRNA and GA1 levels increased, implying that GA20 was being used for de novo synthesis of GA1. The presence of the embryo axis was required for stimulation of cotyledonary GA1 synthesis at the mRNA and enzyme activity levels. As the embryo axis doubled in size, PsGA20ox1 and PsGA3ox1 transcripts increased, both GA1 and GA8 were detectable, PsGA2ox2 transcripts decreased, and PsGA2ox1 transcripts remained low. Cotyledonary-, root-, and shoot-specific expression of these GA biosynthesis genes and the resultant endogenous GA profiles support a key role for de novo GA biosynthesis in each organ during germination and early seedling growth of pea.